Dominant negative mutations in the α-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae

Leavitt, LuAnn M.; Macaluso, C. R.; Kim, K. S.; Martin, Negin P.; Dumont, Mark E.

doi:10.1007/s004380051039

Cited by 45 publications

(126 citation statements)

References 61 publications

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“…Our data and those previously reported (16,17,19) Table 1). Some of the phenotypes of the Ala-scanned Ste2p were similar to those reported in Cys scanning and random mutagenesis of this receptor.…”

Section: Discussionsupporting

confidence: 90%

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Interacting Residues in an Activated State of a G Protein-coupled Receptor

Lee

Naider

Becker

2006

Journal of Biological Chemistry

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Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone ␣-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn 205 as a potential interacting partner with the Tyr 266 residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/ Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.The tridecapeptide ␣-factor pheromone ( 1 WHWLQLKPGQPMY 13 ) of Saccharomyces cerevisiae and Ste2p, its cognate G protein-coupled receptor (GPCR), 3 have been used extensively as models for peptide ligand-GPCR structure and function (1). A major goal of GPCR studies is to ascertain the interactions between ligand and receptor to aid in the identification of analogs used for modulation of receptor activity and to understand how the receptor activates its signal transduction pathway. GPCRs are extremely important for medicine as they represent the target for the majority of prescribed drugs (2).To identify Ste2p residues or regions involved in ␣-factor binding, a number of experiments have been performed. Chimeric receptors between the closely related S. cerevisiae and Saccharomyces kluyveri ␣-factor receptors implicated the involvement of portions of EL1 (extracellular loop 1), EL3, and the N-terminal extracellular region of transmembrane 1 (TM1) in the specificity of ligand recognition (3, 4). Our group using site-directed mutagenesis of Ste2p and binding assays with different ␣-factor analogs suggested that portions of TM1 and TM6 were important for ligand interaction (5) and that the tenth residue of ␣-factor is in close proximity to Ser 47 and Thr 48 in TM1 of Ste2p (6). We found that Tyr 266 i...

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Section: Discussionsupporting

confidence: 90%

“…Affinity of ␣-factor for receptor Biological activity co-expressed receptor (16,17). Plasmids encoding either wild-type or N205A Ste2p were transformed into LM23-3az, a strain containing a chromosomal copy of WT Ste2p.…”

Section: Receptormentioning

confidence: 99%

Interacting Residues in an Activated State of a G Protein-coupled Receptor

Lee

Naider

Becker

2006

Journal of Biological Chemistry

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“…Ser184, a conserved polar residue at the extracellular end of H4, may form an interhelical hydrogen bond with Ser207 on H5. Thr177 and Tyr181 are sites where mutation leads to loss of function (85). Both amino acids are oriented toward the center of the helical bundle between H3 and H5.…”

Section: H4mentioning

confidence: 99%

Comparison of Class A and D G Protein-Coupled Receptors: Common Features in Structure and Activation

et al. 2005

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All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure-function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast α-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors.G protein-coupled receptors (GPCRs) 1 are the largest and most diverse superfamily of membrane receptors. GPCRs are characterized by their common 7 transmembrane (TM) helix architecture and their ability to activate heterotrimeric G proteins. Surprisingly, there is no significant sequence similarity across the GPCR superfamily. In fact, GPCRs have been grouped into at least five distinct classes (1) with no recognizable sequence similarity between the various classes (1,2). Nevertheless, the working assumption has been that these receptors have a common fold and activation mechanism. For example, the amino acid sequences of the yeast pheromone GPCRs, such as the α-factor receptor, diverge considerably from those of the mammalian hormone GPCRs, such as the β-adrenergic receptor. Yet, in both of these receptors, ligand binding occurs within the core of the 7 TM helices and is, in part, mediated by aromatic amino acids on transmembrane helix H6

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“…The shading indicates Cys substitution mutations that caused a significant decrease in receptor function. Red indicates residues affected by dominant-negative mutations (9,25,55). This topographical representation of Ste2p was the working model for the TMDs used at the start of these studies.…”

Section: Cysteine-scanning Mutagenesis Across Extracellular Ends Of Tmentioning

confidence: 99%

“…Analysis of interspecies chimeric receptors first implicated the extracellular regions of Ste2p in ligand binding specificity (44). Genetic screens for dominant-negative STE2 mutants subsequently identified a collection of mutants that implicated 13 different residues that were predicted to be near the extracellular ends of the TMDs in receptor function (9,25,55). These dominantnegative receptors are defective either in binding ␣-factor or in receptor activation in response to ␣-factor.…”

mentioning

confidence: 99%

A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

Lin

Duell

Konopka

2004

Molecular and Cellular Biology

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The ␣-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

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Dominant negative mutations in the α-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae

Cited by 45 publications

References 61 publications

Interacting Residues in an Activated State of a G Protein-coupled Receptor

Interacting Residues in an Activated State of a G Protein-coupled Receptor

Comparison of Class A and D G Protein-Coupled Receptors: Common Features in Structure and Activation

A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

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